5. The test shall be carried out as follows—
A representative 1 kilogram sample of solid mineral hydrocarbon or, if this amount is not available, the entire sample shall be placed in a beaker of a capacity three times the volume of the sample and heated with occasional stirring on a steam bath until the sample and completely melted and homogeneous. Four 25 gram ±0.2 gram portions of the melted sample shall be weighed in separate 100 millilitre beakers. Three of the portions shall be reserved for later replicate analysis as necessary. One weighed portion shall be poured immediately after remelting on the steam bath into a 500 millilitre separatory funnel containing 100 millilitres of the pre-equilibrated sulphoxide-phosphoric acid mixture which has been heated in the heating jacket at a temperature just high enough to keep the sample melted. (Note: In pre-heating the sulphoxide-acid mixture, the stopper of the separatory funnel shall be removed at intervals to release the pressure.)
The transfer of the sample to the funnel in the jacket with portions of the pre-equilibrated iso-octane shall be promptly completed, warming the beaker, if necessary, and using a total volume of just 50 millilitres of the solvent. If the sample comes out of solution during these operations, the stoppered funnel shall be left in the jacket until the sample redissolves. The stopper shall be removed from the funnel at intervals to release pressure. When the sample is in solution, the funnel shall be removed from the jacket and shaken vigorously for 2 minutes. Three 250 millilitre separatory funnels shall be set up, each containing 30 millilitres of pre-equilibrated iso-octane. After separation of the liquid phases, the contents shall be allowed to cool until the main portion of the sample in iso-octane solution begins to show a precipitate. The funnel shall be gently swirled when precipitation first occurs on the inside surface of the funnel to accelerate the process. The lower layer shall be carefully drawn off, filtered through a thin layer of glass wool fitted loosely in a filter funnel into the first 250 millilitre separatory funnel, and washed in tandem with the 30 millilitre portions of iso-octane contained in the 250 millilitre separatory funnels. The shaking time for each wash shall be 1 minute. The extraction operation shall be repeated with two additional portions of the sulphoxide-acid mixture. The funnel shall be replaced in the jacket after each extraction to keep the sample in solution and each extractive shall be washed in tandem through the same three portions of iso-octane.
The successive extractives (totalling 300 millilitres) shall be collected in a 2 litre separatory funnel, containing 480 millilitres of distilled water, mixed, and allowed to cool for a few minutes after the last extractive has been added. 80 millilitres of iso-octane shall be added to the solution and extracted by shaking the funnel vigorously for 2 minutes. The lower aqueous layer shall be drawn off into a second separatory funnel (preferably 2 litre) and the extraction repeated with 80 millilitres of iso-octane. The aqueous layer shall be drawn off and discarded. Each of the 80 millilitre extractives shall be washed three times with 100 millilitre portions of distilled water. The shaking time for each wash shall be 1 minute. The aqueous layers shall be discarded. The first extractive shall be filtered through anhydrous sodium sulphate pre-washed with iso-octane prepared as directed in paragraph 4(1)(j) of this Part of this Schedule into a 250 millilitre Erlenmeyer flask (or optionally into the evaporation flask). The first separatory funnel shall be washed with the second 80 millilitre iso-octane extractive and passed through the sodium sulphate. The second and first separatory funnels shall be washed successively with a 20 millilitre portion of iso-octane, and the solvent passed through the sodium sulphate into the flask. 1 millilitre of n-hexadecane shall be added to the contents of the flask and the iso-octane evaporated on the steam bath under nitrogen. Evaporation shall be discontinued when not more than 1 millilitre of residue remains. A 10 millilitre portion of iso-octane shall be added to the residue and the mixture re-evaporated to 1 millilitre of n-hexadecane. This operation shall be repeated once.
The residue shall be quantitatively transferred with iso-octane to a 25 millilitre volumetric flask, made to volume and mixed. The extinction of the solution in the 4 centimetre path length cells compared to iso-octane as reference between 280 and 400 millimicrons shall be determined, taking care to lose none of the solution in filling the sample cell. The extinction values shall be corrected for any extinction derived from reagents as determined by carrying out the procedure without a solid mineral hydrocarbon sample. If the corrected extinction does not exceed the limits prescribed in paragraph 6 of this Part of this Schedule, the sample shall be deemed to satisfy the requirements of the test. If the corrected extinction per centimetre path length exceeds the limits prescribed, the test shall be continued as follows:
The iso-octane solution shall be quantitatively transferred to a 125 millilitre flask, equipped with 24/29 British Standard 572:1960 joint, and the iso-octane evaporated on the steam bath under a stream of nitrogen to a volume of 1 millilitre of n-hexadecane. 10 millilitres of methyl alcohol and approximately 0.3 gram of sodium borohydride shall be added to the contents of the flask. (In order to minimize exposure of the borohydride to the atmosphere, a measuring dipper may be used.) A water-cooled condenser equipped with a 24/29 British Standard 572:1960 joint and with a drying tube shall be fitted to the flask immediately. The flask shall be shaken until the borohydride is dissolved, and allowed to stand for 30 minutes at room temperature, with intermittent swirling. At the end of this period, the flask shall be disconnected and the methyl alcohol evaporated on the steam bath under nitrogen until the sodium borohydride begins to come out of the solution. 10 millilitres of iso-octane shall then be added and the mixture evaporated to a volume of about 2–3 millilitres. A further 10 millilitres of iso-octane shall be added and the mixture concentrated to a volume of approximately 5 millilitres. The flask shall be swirled repeatedly to ensure adequate washing of the sodium borohydride residues.
The tetrafluoroethylene polymer disc shall be fitted on the upper part of the stem of the chromatographic tube. The tube with the disc shall be placed on the suction flask and the vacuum applied (approximately 135 millimetres mercury pressure). 14 grams of the 2:1 magnesium oxide-diatomaceous earth mixture shall be weighed and the adsorbent mixture poured into the chromatographic tube in approximately 3 centimetre layers. After the addition of each layer, the top of the adsorbent shall be levelled off with a flat glass rod or metal plunger by pressing down firmly until the adsorbent is well packed. The topmost few millimetres of each adsorbent layer shall be loosened with the end of a metal rod before the addition of the next layer. Packing in this manner shall be continued until all the 14 grams of the adsorbent have been added to the tube. The top of the adsorbent shall be levelled off by pressing down firmly with a flat glass rod or metal plunger to make the depth of the adsorbent bed approximately 12.5 centimetres. The vacuum shall then be turned off and the suction flask removed. The 500 millilitre reservoir shall be fitted onto the top of the chromatrographic column and the column pre-wetted by passing 100 millilitres of iso-octane through the column. The nitrogen pressure shall be adjusted so that the rate of descent of the iso-octane coming off the column is between 2–3 millilitres per minute. Pressure shall be discontinued just before the last of the iso-octane reaches the level of the adsorbent. (Caution: The liquid level must not, at any time, recede below the adsorbent level.) The reservoir shall be removed and the 5 millilitre iso-octane concentrate solution decanted onto the column. With slight pressure again the liquid level shall be allowed to recede to barely above the adsorbent level. The flask and the residue shall be thoroughly washed with two 5 millilitre portions of iso-octane which shall be rapidly transferred to the column in a similar manner. Just before the final 5 millilitres wash reaches the top of the adsorbent, 100 millilitres of iso-octane shall be added to the reservoir, and the percolation continued at the 2–3 millilitres per minute rate. Just before the last of the iso-octane reaches the adsorbent level, 100 millilitres of 10 per cent. benzene in iso-octane shall be added to the reservoir, and the percolation at the aforementioned rate continued. Just before the solvent mixture reaches the adsorbent level, 25 millilitres of 20 per cent. benzene in iso-octane shall be added to the reservoir and the percolation continued at 2–3 millilitres per minute until all this solvent mixture has been removed from the column. All the elution solvents collected up to this point shall be discarded. 300 millilitres of the acetone-benzene-water mixture shall be added to the reservoir, and the mixture percolated through the column to elute the polynuclear compounds. The eluate shall be collected in a clean 1 litre separatory funnel. The column shall be allowed to drain until most of the solvent mixture has been removed. The eluate shall be washed three times with 300 millilitre portions of distilled water, shaking well for each wash. (Small amounts of sodium chloride may be added to facilitate separation.) The aqueous layer shall be discarded after each wash. After the final separation, the residual benzene shall be filtered through anhydrous sodium sulphate pre-washed with benzene (prepared as directed in paragraph 4(1)(j) of this Part of this Schedule) into a 250 millilitre Erlenmeyer flask (or into the evaporation flask). The separatory funnel shall be washed with two additional 20 millilitre portions of benzene which shall also be filtered through the sodium sulphate. 1 millilitre of n-hexadecane shall be added and the benzene completely removed by evaporation under nitrogen, using the special procedure to eliminate benzene described in paragraph 4(1)(a)(v) of this Part of this Schedule. The residue shall be quantitatively transferred with iso-octane to a 25 millilitre volumetric flask and adjusted to volume. The extinction of the solution in the 4 centimetre path length cells compared to iso-octane as reference between 250 and 400 millimicrons shall be determined. Correction shall be made for any extinction derived from the reagents, as determined by carrying out the procedure without a solid mineral hydrocarbon sample. If either spectrum shows the characteristic benzene peaks in the 250–260 millimicrons region, the solution shall be evaporated to remove benzene by the procedure described in paragraph 4(1)(a)(v) of this Part of this Schedule. The residue shall be dissolved and transferred quantitatively, and adjusted to volume in iso-octane in a 25 millilitre volumetric flask. The extinction shall again be recorded. If the corrected extinction does not exceed the limits prescribed in paragraph 6 of this Part of this Schedule, the sample shall be deemed to satisfy the requirements of the test.